1. Technical Field
The subject invention relates to isolated nucleic acid sequences or genes involved in polyketide synthase (PKS) biosynthetic pathways. In particular, such pathways are involved in the production of polyunsaturated fatty acids (PUFAs) such as, for example, Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA). Specifically, the invention relates to isolating nucleic acid sequences encoding proteins involved in eukaryotic PUFA-PKS systems and to uses of these genes and encoded proteins in PUFA-PKS systems, in heterologous hosts, for the production of PUFAs such as EPA and DHA.
2. Background Information
Long chain polyunsaturated fatty acids (PUFAs) that contain 20 or 22 carbon atoms (C20-, C22-PUFAs) are essential components of membrane phospholipids and serve as precursors of eicosanoids like prostaglandin, leukotrienes and thromboxanes. They also play a pivotal role in various biological functions such as fetal growth and development, retina functioning and the inflammatory response. The n-6 fatty acids and the n-3 fatty acids are the two major classes of long chain PUFAs. In mammals, the major endpoint of the n-6 pathway is arachidonic acid (ARA, 20:4n-6), and the major endpoints of the n-3 pathway are eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). n-6 and n-3 PUFAs are metabolically and functionally distinct, quite often having opposing physiological functions; thus, their balance is important for homeostasis. An excess of n-6 PUFAs shifts the physiological state to one that is prothrombotic and preaggregratory, leading to inflammatory and cardiovascular complications. On the other hand, n-3 PUFAs such as EPA and DHA have been shown to have therapeutic value in prevention and treatment of diseases such as, for example, cardiovascular disease, inflammation, arthritis and cancer. Thus, there is interest in identifying inexpensive and renewable sources of EPA and DHA.
A large number of lower eukaryotes like fungi and algae produce long chain PUFAs such as EPA and DHA. The exact mechanism of PUFA biosynthesis in these organisms is unknown but is presumed to be similar to that of mammals (i.e., an aerobic pathway involving an alternating series of desaturations and elongations catalyzed by a series of enzymes called desaturases and elongases). Many of these enzymes have already been identified in several of these PUFA-rich fungi such as Thraustochytrium sp., Mortierella sp., etc (Knutzon et al., J. Biol. Chem (1998) 273:29360–29366; Parker-Barnes et al., Proc. Natl. Acad. Sci. USA. (2000) 97:8284–8289; Huang et al., Lipids (1999) 34:649–659; Qiu et al., J. Biol. Chem. (2001) 276:31561–31566).
Recently, Metz et al. (Science (2001) 293: 290–293) proposed that DHA biosynthesis in Schizochytrium, an organism that belongs to the Thraustochytrid family, occurs via a novel polyketide synthase (PKS) pathway rather than the desaturase/elongase pathway (see also U.S. Pat. No. 6,566,583). This mechanism is thought to be similar to that used for EPA/DHA production in prokaryotes like Shewanella (Yazawa, Lipids (1996) 31 Suppl: S297–300) and Vibrio (Morita et al., Biotechnol. Lett. (1999) 21:641–646). In particular, PUFA production is initiated by the condensation between a short chain starter unit like acetyl CoA and an extender unit like malonyl CoA. The C4 acyl chain formed is covalently attached to an acyl carrier protein (ACP) domain of the PKS complex and goes through successive rounds of reduction, dehydration, reduction, and condensation, with the acyl chain growing by C2 units with each round. A novel dehydratase/isomerase has been proposed to exist in the complex (Metz et al., Science (2001) 293:290–293) that can catalyze trans- to cis-conversion of the double bonds, thus generating double bonds in the correct position of EPA and DHA.
The genes involved in the PUFA-PKS pathway have been identified from a number of marine organisms including Shewanella. In Shewanella, these genes were arranged in five open reading frames (ORFs) of ˜20 kb in length and were shown to be sufficient for EPA production when tested in E. coli (Yazawa, Lipids (1996) 31 Suppl: S297–300). Examination of the protein sequences encoded by these five ORFs revealed that at least eleven enzymatic domains could be identified, seven of which were more strongly related to PKS proteins (Metz et al., Science (2001) 293:290–293) rather than to the fatty acid synthase (FAS) proteins that were suggested earlier (Watanabe et al., J. Biochem. (1997) 122:467).
It has been suggested that in Shewanella, at least some of the double bonds are introduced into EPA by a dehydratase-isomerase mechanism catalyzed by the fabA-like domain present in ORF 7 of the Shewanella PUFA-PKS cluster (Metz et al., Science (2001) 293:290–293). Expression studies of the Shewanella PKS gene cluster in E. coli revealed that EPA production could take place in the absence of oxygen indicating that the aerobic desaturase pathway did not play any role in EPA production in these marine bacteria. Thus, PUFA production in this marine bacteria is thought to occur via a novel PKS-like pathway and this is thought to be widespread in marine bacteria that make PUFAs, since genes with high homology to the Shewanella PUFA-PKS gene cluster have been identified in Vibrio marinus (Tanaka et al., Biotechnol. Lett. (1999) 21:939) and in Photobacterium profundum (Allen et al., Appli. Environ. Microbiol. (1999) 65:1710). The PKS pathways for PUFA synthesis in Shewanella and Vibrio marinus have been described in U.S. Pat. No. 6,140,486.
Genes homologous to the Shewanella PUFA-PKS gene cluster were recently identified in Schizochytrium, a marine eukaryote that produces DHA (Metz et al., Science (2001) 293: 290–293; see also U.S. Pat. No. 6,566,583). Labeling experiments with Schizochytrium demonstrated that DHA was produced solely from an acetate precursor, rather than from any C18 fatty acid intermediate, pointing to the PKS-PUFA pathway as being functional in DHA production rather than the aerobic desaturase pathway.
Because of the increased demand for PUFAs such as EPA and DHA, alternate sources of these PUFAs are being sought after. The current natural sources of n-3 PUFAs such as fish oil are not economical or renewable and thus not suitable for commercial needs. Thus, the development of transgenic plant oils enriched with ω-3 PUFAs is currently being considered. For this, the plant will need to be genetically engineered to contain desaturase and elongase genes that are involved in EPA/DHA production. However, this would require expression of six to seven separate enzymes simultaneously in plants, and further manipulations might be necessary to control the flux through the pathway, target these genes to specific organelles, and/or modulate gene expression so as to prevent the accumulation of undesirable intermediates. Thus, it would be of interest to identify alternate PUFA biosynthesis pathways such as the PUFA-PKS pathway.
Although the bacterial PUFA-PKS genes do provide a novel resource for producing transgenic plant oils, it is not known how these bacterial genes will function in a eukaryotic host. Also, the source organisms for these genes grow in cold marine environments and their enzyme systems might not function well at or above 30° C. which could pose a problem for expression in some crops. Additionally, the PUFAs in these marine bacteria are not stored in the triglyceride form since these organisms are not oleaginous strains; thus, the PUFA-PKS system in these organisms cannot direct triglyceride formation. These shortcomings may be overcome by identifying additional PUFA-PKS genes from eukaryotic sources that make triglycerides. The identification of a PUFA-PKS gene cluster from Schizochytrium, fits this criteria. However, the amount of DHA produced by Schizochytrium is low compared to other Thraustochytrid species, and a large fraction of this DHA is found in the phospholipid fraction rather than in the triglyceride form (Kendrick et al., Lipids (1992) 27:15–20). Therefore, there is a need to identify other PUFA-PKS systems from eukaryotes that produce large amounts of DHA that is found in the triglyceride fraction, as well as EPA. Thraustochytrium aureum is an ideal candidate since this organism belongs to the same Thraustochytrid family as Schizochytrium does, but produces copious amounts of DHA (˜30% of the total lipid is DHA) as compared to Schizochytrium, and has a major portion of its DHA in the triglycerol fraction (Kendrick et al., Lipids (1992) 27:15–20). Identification of the PUFA-PKS system from Thraustochytrium aureum provides an excellent alternative for the production of PUFA-enriched transgenic oils.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.